2-Dimensional ultra-high performance liquid chromatography and DMT-MM derivatization paired with tandem mass spectrometry for comprehensive serum N-glycome characterization

Glycosylation is a prominent co- and post-translational modification which contributes to a variety of important biological functions. Protein glycosylation characteristics, particularly N-glycosylation, are influenced by changes in one's pathological state, such as through the presence of disease, and as such, there is great interest in N-glycans as potential disease biomarkers. Human serum is an attractive source for N-glycan based biomarker studies as circulatory proteins are representative of one's physiology, with many serum proteins containing N-glycosylation. The difficulty in comprehensively characterizing the serum N-glycome arises from the absence of a biosynthetic template resulting in great structural heterogeneity and complexity. To help overcome these challenges we developed a 2-dimensional liquid chromatography platform which utilizes offline weak anion exchange (WAX) chromatography in the first dimension and hydrophilic interaction liquid chromatography (HILIC) in the second dimension to separate N-glycans by charge, corresponding to degree of sialylation, and size, respectively. Performing these separations offline enables subsequent derivatization with 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) for sialic acid linkage determination and the identification of sialic acid linkage isomers. Subsequent tandem mass spectrometry analysis revealed the identification of 212 complete and partial N-glycan structures including low abundant N-glycans containing acetyl and sulphate modifications. The identifications obtained through this platform were then applied to N-glycans released from a set of stage 3 gastric cancer serum samples obtained from patients before (pre-op) and after (post-op) tumour resection to investigate how the serum N-glycome can facilitate differentiation between the two pathological states.